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Analysis of genes induced in human periodontal ligament cells respond to the mechanical stress

分析人牙周膜细胞对机械应力反应诱导的基因

基本信息

批准号：
12470460
负责人：
TAKANO Teruko
金额：
$ 8.83万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470460/
关键词：
periodontal ligament mechanical stress Micro Array マイクロアレイ法

项目摘要

Human PDL tissue was excised from a premolar or a third molar, which were extracted from orthodontic reasons, and placed onto flexible bottom plate with Dalbecco's modified eagle medium (DMEM) containing 10 % fetal bovine serum. After 10 to 14 days fibroblastic cells were extended and after 1 or 3 months later, tension force was applied on the primary culture cells for 24h. The cells derived from the PDL tissue from the same patient without stress were used for control. Total RNA was extracted from both cells and reverse transcribed into cDNA, followed by PCR. The difference of the expression level of 1176 kinds of genes was analyzed by Micro Array method. Same experiments were done with other 3 patient's periodontal tissue and mRNA was collected at some points from early stage to late stage after mechanical stress. The genes reinforced by the mechanical stress, and those suppressed were identified. Some genes expressed strongly in early stage transiently, that decreased with time, and some showed reverse pattern. On the other hand, the mechanical tension force was applied using the cells that were subcultured to 3 passages. The results were almost the same to the experiments using the primary culture cells, although the number of genes responded to the stress were less and the amount of the expression change by stress was smaller. These results suggested that the aging accompanied with the long culture days or subculture lowed down the sensitivity for the mechanical stress or caused the selection for the cells that cannnot respond to the stress.Now, we are preparing the paper about these observations to submit to the international journals, and some of them have been accepted.

从因正畸原因提取的前磨牙或第三磨牙中切除人 PDL 组织，并将其放置在含有 10% 胎牛血清的 Dalbecco 改良 eagle 培养基 (DMEM) 的柔性底板上。 10至14天后，成纤维细胞延伸，1或3个月后，对原代培养细胞施加张力24小时。源自同一患者无应激的 PDL 组织的细胞用于对照。从两个细胞中提取总RNA并反转录成cDNA，然后进行PCR。采用Micro Array方法分析1176种基因表达水平的差异。对另外3名患者的牙周组织进行了相同的实验，并在机械应力后从早期到晚期的某些时间点收集了mRNA。机械应力增强和抑制的基因被鉴定出来。有些基因在早期短暂强烈表达，随着时间的推移而减弱，有些则表现出相反的模式。另一方面，使用传代培养至3代的细胞施加机械张力。结果与使用原代培养细胞的实验几乎相同，尽管响应应激的基因数量较少并且应激引起的表达变化量较小。这些结果表明，随着培养时间长或继代培养而发生的老化降低了对机械应力的敏感性，或导致细胞选择出不能响应应力的细胞。目前，我们正在准备将这些观察结果提交给国际期刊的论文，其中一些已被接受。